Preparation of enzymatically active membranes

ABSTRACT

1. A PROCESS OF PRODUCING AN ENZYMATICALLY ACTIVE MEMBRANE WHICH COMPRISES SWELLING A MEMBRANCE FORMED FROM A PROTEIN SELECTED FROM THE GROUP CONSISTING OF COLLAGEN, ZEIN, CASEIN, OVALBUMIN, WHEAT GLUTEN, FIBRINOGEN, MYSOIN, AND MUCOPROTEIN, OR A POLYPEPTIDE SELECTED FROM THE GROUP CONSISTING OF POLYGLUTAMATE, POLYASPRTATE, POLYPHENYLAMINE, POLYTRYROSINE AND COPOLYMER OF LEUCINE AND P-AMINO PHENYLANALINE TO ITS MAXIMUM CAPACITY, AND SOAKING SAID SWOLLEN MEMBRANE IN AN AQUEOUS SOLUTION OF AN ENZYME AT A TEMPERATURE OF 4-10*C. FOR A PERIOD OF 10 HOURS TO 2 DAYS UNTIL SAID ENZYME COMPLEXES BY FORMING COMPLEXING GROUPS SELECTED FROM THE GROUP CONSISTING OF MULTIPLE HYDROGEN BONDS, SALT LINKAGES AND VAN DER WAALS INTERACTIONS WITH SAID MEMBRANE TO FORM AN ENZYME MAMBRANE COMPLEX, AND THEREAFTER DYING SAID ENZYMEMEMBRANE COMPLEX.

United States Patent 3,843,446 PREPARATION OF ENZYMATICALLY ACTIVEMEMBRANES Wolf R. Vieth, Belle Mead, Shaw S. Wang, New Brunswick, andSeymour G. Gilbert, Piscataway, N.J., assignors to Research Corporation,New York, N.Y. No Drawing. Filed Apr. 20, 1971, Ser. No. 135,753

Int. Cl. C07g 7/02 U.S. Cl. 195-68 4 Claims ABSTRACT OF THE DISCLOSUREEnzymatically active protein-enzyme complex membranes are prepared bytreating a swollen protein membrane with an aqueous solution of acompatible active enzyme. These membranes are used to effect enzymaticreactions.

BACKGROUND OF THE INVENTION Field Of The Invention Description Of ThePrior Art Enzymes are protein catalysts which have been used for a widevariety of industrial and research applications, particularly inpharmaceuticals, paper and textile process ing, etc. They are highlyspecific in their activity and enerally do not generate significantquantities of undesirable byproducts. Enzyme reactions are industriallyadvantageous since they do not require a large investment in heattransfer equipment and can be easily staged, thereby minimizing theproblems associated with interstage product separations.

One problem which has long concerned those dealing with industrialapplications of enzymes, however, is the difficulty in separating orrecovering enzyme materials. In most commercial processes, the enzymaticreaction is effected by simply admixing the enzyme with the substrate,and thereafter inactivating and/or recovering the enzyme from theproducts or the unreacted substrate following the reaction. Thisprocedure, however, has frequently resulted in damage to the product andinherent loss of large quantities of enzyme, since usually no enzyme isrecovered or, if attempted, the yields are quite low.

Another problem which has been of significant concern to those engagedin this technology, is that the enzymes usually are used in an aqueousdispersion form. As a rule, however, enzymes in this form have a limitedshelf life and, especially, if stored in dilute form, will undergo rapidloss of activity upon storage.

To alleviate these problems, the art has developed various so-calledimmobilized enzymes in which the enzymes are immobilized or bound toinert or insoluble carriers. At the completion of the enzymaticreaction, these insoluble enzyme-containing materials can be separatedfrom the unreacted substrate or product by techniques such asultrafiltration or the like.

The selection of a suitable inert carrier, however, has been quitedifficult, since the carrier must not only be inert to the enzyme, butit must not inhibit the catalytic activity of the enzyme, nor causeundesirable unspecific adsorption. Moreover, the carrier should presenta minimum of steric hindrance toward the enzyme-substrate reaction. Awide variety of prior art carriers have been 3,843,446 Patented Oct. 22,1974 "ice proposed, depending upon the particular type of enzyme usedand the particular enzymatic reaction desired. For instance, among thoseprior art carriers disclosed in the open literature include, syntheticpolymers such as polyamides, cellulose derivatives, various clays, andion-ex change resins, particularly DEAE-cellulose, and DEAE- dextrans,as discussed in Suzuki, et al., Agr. Biol. Chem, Volume 30, No. 8, pages807-812 (1966). Prior art methods of preparing immobilized enzymes haveincluded direct covalent bonding, indirect bonding through anintermediate compound, crosslinking of the enzyme or trapping the enzymein polymer lattices.

None of these prior art techniques or carriers, however, have beenentirely satisfactory for all purposes. Synthetic polymer carriers areexpensive and frequently are not readily available. Moreover, they oftenrequire special treatment in order to chemically bind the enzyme to thecarrier. The cellulose derivatives are generally unsuitable as bindersfor carbohydrases, since carbohydrates are substrates for these enzymes.Ion-exchange resins, such as DEAE-cellulose and DEAE-dextran, haveion-exchange properties, which may not be desirable for certainapplications. The problem of enzyme liberation from a carrier is oneweak point in many immobilized enzyme preparations, and is particularlytroublesome in the case of amylase bound to acid clay, which becomesliberated during the hydrolytic reaction of starch.

One particular disadvantage of the prior art methods of immobilizingenzymes is that they have resulted in the formation of insoluble pastes,particles or granular materials. While such forms are suitable, and evenpossibly desirable for certain applications, for other applications,these forms impose severe limitations, especially when they are used forlarge-scale or long-term continuous processes.

A need exists, therefore, for an enzyme carrier which can be formed intoa variety of shapes and hence can be used as a structural part of areaction system, so as to eliminate entirely separation problems. Morespecifically, a need exists for a membrane or film-like carrier which iscapable of complexing and binding enzymes thereto without hinderingtheir catalytic activity, so that enzymatic reactions can be effectedmerely by passing the substrate over the active membrane or film. Thepresent invention fills such a need.

SUMMARY OF THE INVENTION Accordingly, one object of this invention is toprovide immobilized enzymes in membrane form.

Another object of this invention is to provide a technique for producingimmobilized enzymes in membrane form.

A still further object of this invention is to provide a technique foreffecting enzymatic reactions by passing an enzymatically activesubstrate over an enzyme-carrier complex membrane, which ischaracterized by good catalytic activity.

Briefly, these and other objects have now been attained in one aspect ofthis invention by immobilizing enzymes on protein membranes.

Suitable membranes include both synthetic polypeptides and naturalprotein, in unmodified or modified forms. Enzyme immobilization isaccomplished in one case by swelling a protein membrane, and thereaftersoaking said membrane in an aqueous dispersion of an enzyme for a periodof time sufficient to complex the enzyme with the protein.

The complexing mechanism between enzymes and protein membranes orfilm-like protein carriers involves the formation of multiple hydrogenbonds, salt linkages, and van der Waals interactions. Complex formationis fatcilitated at a pH between the isoelectric points of the enzyme andthe protein membrane.

DESCRIPTION OF THE PREFERRED EMBODIMENTS A Wide variety of syntheticpolypeptides and natural proteins may be used in the present invention.Non limiting examples of suitable natural proteins include collagen,zein, casein, ovalbumin, wheat gluten, fibrinogen, myosin, mucoprotein,and the like. Nonlirniting examples of suitable synthetic polypeptidesinclude polyglutamate, polyaspartate, polyphenylalanine, polytyrosine,and copolymers of leucine with p-amino phenylaniline.

The selection of a particular synthetic polypeptide or natural protein,in modified or unmodified form, will be largely determined by the natureof the enzyme being complexed, the substrate to be treated, and thereaction environment to be encountered. Because of their inertness to alarge number of enzymes, collagen and zein are preferred natural proteinmaterials. While the following description of this invention illustratesthe use of collagen and zein, it will be apparent that the invention isequally applicable, with obvious modifications, to other membranes ofthe aforementioned types. In one embodiment, a collagen film is preparedby casting a dispersion of collagen according to state of the arttechniques. The film is then swollen, washed in water and soaked in anenzyme solution. After refrigerated storage to allow diffusion of theenzyme into the collagen, the film may be layered on a base, such as acellulose acetate film, and dried.

Collagen is a hydroxyproline, glycine-type protein, which is the chieforganic constituent of connective animal tissue and bones. Chemically,collagen is distinguishable from other proteins by its unusually highglycine content, which accounts for approximately one-third of the aminoacid residues therein; the high content of proline and hydroxyproline;the presence of hydroxyglycine, which is unique among proteins; and inhaving notably small amounts of aromatic and sulfur-containing aminoacids. It can be obtained in good yields from a wide variety of mammaland fish parts, and is frequently obtained from pork, sheep and beeftendons; pigskins; tanners stock, which are calfskins not usable forleather; and ossein, which is tissue obtained by drying cattle bonesremaining after acid treatment to remove calicum phosphate.

One suitable method for forming a collagen membrane is as follows: Thecollagen source is first treated with an enzyme solution to dissolve theelastin which encircles and binds the collagen fibers. Proteolyticenzymes, from either plant or animal sources, may be used for thispurpose, although other types of enzymes are equally satisfactory. Thecollagen source is then washed with water and the soluble proteins arelipids are removed by treatment with a dilute aqueous solution of achelating agent, such as ethylene diamine tetrasodium tetraacetate. Thecollagen fibers are then swollen in a suitable acid, such as cyanoaceticacid, as described in Hochstadt, et al., US. Pat. 2,920,000, so as toform a collagen fiber dispersion. This dispersion can then be extrudedor cast into a suitable membrane form. The dried collagen membrane isthen annealed at 60 C., 95% RH. for 48 hours. The said collagen fiberdispersion can also be electrodeposited according to British Patent1,153,551 to form suitable membranes.

Of course, any one of the many state of the art techniques can be usedto form suitable collagen membranes, and the above descriptions are onlyexemplary of suitable prior art techniques.

The collagen membranes useful in the present invention generally have athickness of from 0.005 mm. to 0.1 mm. and preferably from 0.01 mm. to0.05 mm. When the thickness is less than 0.005 mm., the membrane losesits desirable strength and may not form a completely integral filmwithout pinholes or other structural defects.

When the thickness exceeds 0.1 mm., the cost of the complex increaseswithout necessarily increasing the efficiency of the complex in itsperformance.

Other materials may be added to the membrane to accomplish specificaims. For example, plasticizers may be used to modify the molecularstructure of the membrane to provide greater resilience by allowing forchain slippage. Humectants may maintain a more favorable water bindingcapacity. Cross-linking agents, heat annealing, or tanning with chromeor formaldehyde, as described in the prior art, may be employed toinhibit hydrolysis or to provide additional bonding sites for thedesired enzyme, thereby enhancing enzyme retention.

The collagen membrane is then prepared for complexing with the enzyme,generally by being swollen with a low molecular weight organic acid, orin some instances with suitable bases so that the pH ranges from about2-12. Suitable acids include lactic acid and cyanoacetic acid. Ifdesired, plasticizers or other additives heretofore mentioned may beadded during the swelling step. Swelling is accomplished by submergingthe membrane in the acid bath for between A. hour and 1 hour, dependingupon the particular conditions of the bath, generally at roomtemperature in excess of this level will result in the conversion of thecollagen toa soluble gelatin.

The membrane is swollen by the acidity of the organic acid added and theuse of the acid as a plasticizer. No other additive is needed. A changein water binding capacity results from the acid treatment.

Following the swelling treatment, the swollen collagen membrane iswashed thoroughly with water until the pH level of the membrane iswithin the acceptable range for the particular enzyme being complexed.

The swollen, washed membrane is then soaked in an aqueousenzyme-containing solution until complexing occurs. Usually, thisrequires a period of from 10' hours to 2 days. The temperature rangeduring this time should be maintained within 4 C. to 20 0., dependingupon the particular enzyme used. Maximum enzyme uptake is measured byactivity after washing, and indicates when complexing is complete.

The enzyme-collagen complex medium should be carefully dried, preferablyat about room temperature or below, so as not to damage the boundenzyme.

As a second example of using natural protein to complex enzymes, zeinfilm is prepared by casting a solution of zein according to state of theart techniques. The same procedure was used to prepare protein-enzymecomplexes as was done with collagen film, except that the swelling ofthe zein film was aided by adding plasticizers, such as1,5-pentane-diol.

Zein is the prolamin (alcohol-soluble protein) of corn. It is the onlycommercially available prolamin and one of the few readily availableplant proteins. Zein occurs primarily in the endosperm of the cornkernel. The amount of alcohol-soluble protein is directly related to thetotal endosperm protein content, with zein contents ranging from 2.2 to10.4% of the dry substance in various corn samples.

Zein is characterized by a relative deficiency of hydrophilic groups incomparison with most proteins. In fact, the high proportion of nonpolar(hydrocarbon) and acid amide side chains accounts for the solubility ofzein in organic solvents and its classification as a prolamin.

One of the commercial zeins is Argo Zein G200, manufactured by CornProducts Refining Company, Argo, Illinois. Film casting solutions can beformulated on a pure component basis, taking into account the water content of the raw zein and other reagents. The casting solutions areprepared by dissolving the protein in the organic solvent of choice bygentle stirring, at room temperature, for a period of 12 hours, duringwhich period solution is complete. Examples of suitable solvents whichmay be employed include 81% (wt/wt.) isopropyl alcohol and 4% methylcellosolve (ethylene glycol monoethyl ether). The clear solutions, whichcontained 20-30% by weight of dry zein, are of amber color. Curingagents, such as formaldehyde, and a plasticizer may be added shortlybefore film casting.

Of course, any one of the many state of the art techniques can be usedto form suitable zein membranes, and the above description is onlyexemplary of one suitable prior art technique.

The zein membranes useful in the present invention generally have athickness of from 0.005 mm. to 0.1 mm. The zein membrane is thenprepared for complexing with the enzyme by swelling with a plasticizer,if plasticizer was not added before film casting. Suitable plasticizersinclude 1,5-pentane-diol, glycerol, and sorbitol. This is accomplishedby submerging the membrane in a bath of 2% (w./w.) plasticizer in waterfor hours at room temperature. The swollen membrane is then dried withtissue paper and soaked in an aqueous enzyme solution until complexingis completed. Usually, this requires a period of from 10 hours to 2days. The temperature range during this period should be maintainedwithin 4 C. to C., depending upon the particular enzyme used.

The enzyme-zein complex medium should be carefully dried, preferably atabout room temperature or below, so as not to damage the bound enzyme.

A wide variety of different types of enzymes can be complexed withnatural proteins such as collagen, zein, and the like in this manner,depending upon the particular application intended. For instance,suitable enzymes include amylases, lysozyme, invertase, urease,celluloses, catecholmethyltransferase, sucrose 6-glucosyl-transferase,carboxyl esterase, aryl esterase, lipase, pectin esterase, glucoamylase, amylopectin-1,6-glucosidase, oligo-l,6-glucosidase,polygalacturonase, a-glucosidase, fi-glucosidase, B- galactosidase,glucose oxidase, galactose oxidase, catechol oxidase, catalase,peroxidase, lipoxidase, glucose isomerase, pentosanases, cellobiase,xylose isomerase, sulphite oxidase, ethanolamine oxidase, penicillinase,carbonic anhydrase, gluconolactonase, S-keto steroid A 'dehydrogenase,ll-p-hydroxylase, and amino acid acylases. Compatible combinations ofenzymes, and multienzyme systems can also be complexed with the collagenin this manner.

Especially suitable, however, are lysozyme, invertase, urease andamylases. Lysozyme is widely used to hydrolyze microorganisms inpharmaceutical research, and in sewage treatment, either alone or incombination with other enzymes, and/or bacteria. One particularlyimportant application for lysozyme-protein membrane complex is in thelysis of cells.

Invertase or fi-D-fructofuranosidase is widely used in the food andbeverage industries, as well as for analytical purposes. Invertase canbe used to catalyse the hydrolysis of sucrose to glucose and fructose orinvert sugar. Invertase is effective in the hydrolysis offi-D-fructofuranosyl linkages in sucrose, rafiinose, gentianose, andmethyl and 18-fructofructose, One particularly important application foran invertase-protein membrane complex is in the continuous hydrolysis ofsucrose.

Urease is a highly specific enzyme which can catalyze the transformationof urea to ammonium carbonate, and is often used to determine the ureacontent in urine specimens. Because of its highly specific activity, oneutility for the urease-protein complex membrane is in kidney machineapplications. More particularly, urease-protein complex membranes can beused for repeated hydrolysis of urea, such as in the treatment of humanwastes.

et-amylase is referred to as the liquifying enzyme and is known torandomly hydrolyze starch, glycogen, and dextrans. it-amylase canproduce maltose from sugar, glycogen and dextran. Other suitableamylases include tx-glucosidase, amylglucosidase, amylo-l,6-ot-glucosidase (debranching enzyme), oligo-l, 6-glucosidase (limitdextrinase), isomaltase, and isotriase. As used herein, the term amylaserefers generically to one or more of these and other amylases. Oneparticularly important application of the amylase-protein complex of thepresent invention is in the continuous passage of starch substrates overthe enzymatically active membrane to effect continuous hydrolysis ofstarch.

Several enzymes can be simultaneously complexed with the proteinmembrane. For instance, it is quite desirable to complex a-amylase withother types of enzymes, since a-amylase is capable of randomly cleavinga starch molecule, so as to provide reactive sites for other morespecific enzymes.

Immobilized complexes formed in this manner provide good enzymaticactivity. When an enzymatically active substrate is contacted with suchcomplexes, a constant amount of the enzyme remains bound to the carrierthroughout the reaction period so that there is no necessity to providea separate separation procedure, as in the prior art. Moreover, it hasbeen found that the enzyme-protein complexes of the present inventionare stable over long periods of storage and can be washed repeatedlywithout significant loss in enzymatic activity.

While not wishing to be bound by any theory, it is believed that thecomplexing mechanism between protein membranes or film-like proteincarriers and enzymes involves the formation of multiple hydrogen bonds,salt linkages and van der Waals interactions. Complex formation isfacilitated at a pH between the isoelectric points of the enzyme and theprotein membrane.

It should be clearly understood that the art of preparing the membranesfrom collagen films, of the type which are used herein for complexingwith the various enzymes is a well developed art and a variety of stateof the art techniques are available. For instance, the Hochstadt, U.S.Pat. 2,920,000, mentioned above, is merely representative of suitabletechniques for preparing collagen type films and membranes.

Having now generally described the invention, a further understandingcan be obtained by reference to the following examples, which arepresented for purposes of illustration only and are not intended to belimiting unless so specified.

Example 1 (lysozyme) This example demonstrates the formation and use ofa lysozyme collagen membrane complex.

One cc. of a 1 mil thick collagen film (post-heated at 55 C., R. H. for48 hours) was swollen in a lac ic acid solution (pH=3) and then washedin running tap water for 5 minutes. The washed film was then soaked inan enzyme solution of 250 mg. in lysozyme in 15 cc. of water, and storedat 2 C. for 14 hours. The soaked film was then layered on celluloseacetate, and dried at room temperature to yield a lysozyme-collagenmembrane complex.

A solution of Micrococcus lysoa'eictikus (300 mg. of dried cells perliter) was used to assay the enzymatic activity of the complex bymeasuring the decrease in optical density at 450 m of the bacterialsolution. The dried membrane complex was first washed with 10 liters ofrunning water and its initial enzymatic activity measured. This wasdetermined on the basis of the decrease in optical density divided bythe initial optical density after a reaction period of 30 minutes. Thecomplex was washed with 2 liters of water between individualexperiments. Initial activity was 0.538, corresponding to 53.8% of thecells being lysed. The second repetition gave an activity of 0.420,corresponding to 42% lysis; the third repetition gave an activity of0.489, or 48.9% lysis; and the fourth experiment gave an activity of0.533, or 53.3% lysis.

Example 2 (invertase) This example describes the preparation and use ofan invertase-collagen complex. 1.5 cc. of a 1 mil thick collagen film(untanned and stored at room temperature for at least 2 months) wasswollen in a lactic acid solution (pH=3). The film was then washed inWater for onehalf hour, and the washed film was soked in a solution of 140 mg. invertase in 10 cc. of water and stored in a refrigeratorovernight. The soaked film was then layered on a cellulose acetatesubstrate, and dried at room temon a collulose acetate substrate, anddried at room temperature.

'Prior to use, the membrane-urease complex was washed with liters ofwater and stored at 2 C. for 5 days before testing for urease activity.

TABLE 2 Hydrolysis of urea by urease-collagen membrane complex Percentof urea hydrolyzed in the reaction time indicated 1 In units of 3.3X- M(initial). a One cc. membrane complex was used in all the experiments tohydrolyze 50 cc. urea solution.

b Membrane complex used on Day 4 was soaked in 50 cc. of enzyme solution(100 mg. Urease 50 cc.) for 14 hours, and washed with 2 liters waterbefore it was used on Day 5.

6 Average of two experiments.

perature. The dried film, which is a collagen-invertase membranecomplex, was used in the following experiment to hydrolyze sucrose.

400 cc. of 6% sucrose solution was used as a substrate in the enzymeassay. Enzymatic activity was followed polarimetrically in arecirculation reactor system. After the complex was assayed for itsactivity, it was washed with 2 liters of water, and the activity againmeasured. This process was repeated over times, with total wash ings ofover 40 liters. The enzymatic activity of the invertase-collagen complexdecreased gradually after washing, finally reaching a stable limit whichheld constant for over 10 liters of Washing. The total elapsed time atthis stage was 13 days.

TABLE 1 Percent sucrose invention in 30 minutes of reaction time Theenzymatic activity of the invertase complex at the stable lower limitcorresponds to a reaction rate of 1.73 1() mole/liter/ minute. Assigningthe complex the same turnover number as the free enzyme (370 moles ofsucrose per mole of enzyme per second), the reaction rate abovecorresponds to the activity of 21.4 mg. of invertase in 1 liter of a 6%sucrose solution. Since only 1.5 cc. of an invertase complex was used tohydrolyze 400 cc. of substrate, the amount of invertase bound to 1.5 cc.is calculated to be 8.56 mg, or 5.7 mg. of invertase per cc. of thecomplex.

This series of experiments was done over a period of 13 days. Thecomplex was stored at 2 C. in 5 cc. of distilled water when not beingused.

Example 3 (urease) This Example describes the preparation and use ofurease-collagen membranes. One cc. of a 1 mil thick collagen film (postheated at 60 C., 95% RH. for 48 hours) was swollen in lactic acidsolution, pH=3, then washed in water for one-half hour. The washed filmwas then soaked in an enzyme solution of 200 mg. of urease in 15 cc. ofwater for 16 hours. The film was then layered Urease activity wasfollowed by direct potentiometric measurement of ammonium ionconcentration through the use of a Beckman 39137 cationic sensitiveelectrode, and also measured colorimetrically with Nesslers reagent.Ammonium sulfate was used to obtain a standard curve in both cases. Onedrop of freshly prepared Nesslers reagent was added to 2 ml. ofsubstrate solution, and the color intensity was measuredspectrophotometrically at a wavelength of 430 m Three concentrations ofurea, namely, 3.3Xl0" 3.3Xl0' and 3.3 l0- molar, in distilled water wereused. Activity of the membrane complex was tested over a period of oneweek. When not in use, the complex was stored immersed in 50 cc.distilled water at room temperature. Results show that 99% hydrolysis of50 ml. of a 3.3X1O' molar solution was obtained in 20 hours by using 1cc. of the membrane complex, which had already been used for over 1week.

Example 4 (amylase) This Example demonstrates the formation and use ofan amylase-collagen membrane complex. Two cc. of a 1.5 mil thickcollagen film (post-heated at 60 C., RH. for 48 hours) was swollen in alactic acid solution for one-half hour at pH 3. The film was then washedin running tap water for 5 minutes, and the washed film then soaked in asolution of 200 mg. of malt amylase in 15 cc. of water, and stored at 2C. for 15 hours. There was no necessity to remove excess enzymesolution, and the soaked film was immediately layered on a celluloseacetate film substrate 1 to 2 mils thick and dried at room temperature.The dried film, a collagen-amylase membrane complex, was used tohydrolyze starch. 50 cc. of a 1% starch solution was used as a substratein the enzyme assay. Enzymatic activity was measured by the decrease inthe blue color intensity of the starch-iodine complex (change in opticaldensity at 400 Ill 1..) A standard iodine reagent was prepared bydissolving 30 grams of potassium iodide and 3 grams I in one liter ofdistilled water. One drop of this reagent was added to 20 cc. of thereacted substrate which had been incubated with 2 cc. of theamylase-collagen membrane complex at 40 C. for 15 minutes, and theabsorption at 400 m was measured.

The following shows the results of starch hydrolysis by acollagen-amylase membrane complex which was washed with 10 liters ofwater prior to the experiment. After 15 minutes, the color of theiodinated starch solution changed from blue to violet, consistent withthe degradation of the starch molecules from an initial average degreeof polymerization in excess of 30 to a final average degree ofpolymerization of 10 to 15. Upon the first run, amylase activity of 2cc. of film in 50 cc. of a 1% starch solution reacted for 15 minutes at40 C. gave an amylase activity, as measured by the quotient of thedecrease in optical density at 400 mu. divided by the initial opticaldensity at 400 mg, of 0.700. The complex was washed with two liters ofwater, and the second run gave an amylase activity of 0.325. After anadditional washing, an amylase activity of 0.500 was obtained for thethird run.

Example (cellulase) This Example describes the preparation and use of acellulase-collagen complex. 1 cc. of a 1.5 mil thick collagen film(post-heated at 55 C. and 95% RH. for 30 hours, and then stored at roomtemperature for 2 months) was swollen in a lactic acid solution (pH=3).The swollen film was then washed in water for one-half hour, and thewashed film was bathed in a solution of 200 mg. cellulase in cc. ofwater and stored at 4 C. for 14 hours. The soaked film was then layeredon a cellulose acetate substrate, and dried at room temperature. Thedried film was washed in one liter of water for two weeks at 4 C. beforeit was used in the following experiment to hydrolyze carboxyl methylcellulose (CMC). 50 cc.

of 1.5% CMC was used as a substrate in the enzyme assay. Enzymaticactivity was followed by monitoring the decrease in relative viscosityof the substrate, as measured by the Ostwald viscosimeter. Between runs,the film was washed with 2 liters of water. The results obtained areshown in Table 3.

As shown in the above results, the cellulase-collagen complex was ableto decrease the viscosity of the substrate to less than half of itsinitial value after five runs. During the fifth run, the relativeviscosity of the sub strate decreased to 1.43 after 18 hours of reactiontime.

Example 6 (invertase-zein) This Example describes the preparation anduse of an invertase-zein complex. 2 cc. of a 1.5 mil thick zein film(formaldehyde-tanned) was swollen in 2% (W/W) 1,5- pentanediol. The filmwas then dried with tissue paper, soaked in an enzyme solution of 200mg. invertase in cc. of water, and stored in a refrigerator overnight.The soaked film was then layered on a cellulose acetate substrate, anddried at room temperature. The dried film, which is a zein-invertasemembrane complex, was used in the following experiment to hydrolyzesucrose.

400 cc. of 6% sucrose solution was used as a substrate in assayingenzyme activity which was followed polarimetrically in a recirculationreactor system, as in Example 2. After the complex was assayed for itsactivity, it was washed with 2 liters of water and reused. The resultsobtained are shown in Table 4.

TABLE 4 Percent sucrose inverted in 2 hours of reaction No. of washings:time, at 25 C. and pH 6 T0 The enzymatic activity of the invertase-zeincomplex decreased gradually after washing, finally reaching a stablelimit as did the collagen-invertase complex in Example 2.

Example 7 (glucose isomerase) This Example demonstrates the formationand use of a glucose isomerase-collagen membrane complex.

One cc. of a 1 mil thick collagen film (post-heated at 60 C., R. H. for28 hours) was swollen in a lactic acid solution (pH=3) and then washedin running tap Water for 5 minutes. The washed film was then soaked at 2C. for 16 hours in 55 cc. of enzyme solution which had a total enzymaticactivity of 5.4 10- Unit (1 unit=1 mole product formed/ minute.)

The soaked film was then layered on cellulose acetate, and dried at roomtemperature to yield a glucose isomerase collagen membrane complex.

The membrane complex was used to catalyze the isomerization of D-glucosein 50 ml. of a 9% solution buttered at pH 7.2, at 60 C. After a reactiontime of two hours, 1 ml. of the reaction solution was sampled and D-fructose formed was determined by the cysteine-carbazole method. [Z.Dische and E. Borenfreund, J. Biol. Chem., 192 583 (1951).]

The initial activity of the membrane complex was 8.3 10- unit. Themembrane complex was then washed with 1.5 liters of water and stored incc. of water at 4 C. for 17 hours before the second run. The second runshowed a slight increase in activity which was probably due to anincrease in reaction temperature from 60 to 65 C. in this run. An enzymeactivity of 9.8 10* unit was obtained. The membrane complex was thenwashed with another 1.5 liters of water and used in a third run at 60 C.The enzyme activity obtained was 8.7 X 10- unit. By the third run, themembrane complex had been at 60 C. for more than six hours. Theseresults demonstrate the stability and reusability of the membranecomplex.

It will be appreciated that while the foregoing disclosure relates toonly preferred embodiments of the invention for preparing activeinsoluble protein-enzyme complexes, numerous modifications oralterations may be made by those skilled in the art without departingfrom the spirit and scope of the invention as set forth in the appendedclaims.

What is claimed and intended to be secured by Letters Patent of theUnited States is:

1. A process of producing an enzymatically active membrane whichcomprises swelling a membrane formed from a protein selected from thegroup consisting of collagen, zein, casein, ovalbumin, wheat gluten,fibrinogen, mysoin, and mucoprotein, or a polypeptide selected from thegroup consisting of polyglutamate, polyaspartate, polyphenylamine,polytryrosine and copolymer of leucine and p-amino phenylanaline to itsmaximum capacity, and soaking said swollen membrane in an aqueoussolution of an enzyme at a temperature of 410 C. for a period of 10hours to 2 days until said enzyme complexes by forming complexing groupsselected from the group consisting of multiple hydrogen bonds, saltlinkages and van der Waals interactions with said membrane to form anenzyme membrane complex, and thereafter drying said enzymemembranecomplex.

2. The process of Claim 1, wherein said enzyme is selected from thegroup consisting of oxidoreductases, transferases, hydrolases,isomerases, and compatible mixtures thereof.

3. The process of Claim 1, wherein said enzyme is selected from thegroup consisting of lysozyme, urease, amylase, invertase, cellulase,glucose isomerase, and compatible mixtures thereof.

11 12 4. The process of Claim 1, wherein said membrane is Silman, etal., Some Water-Insoluble Papain Derivaswollen atapH of 2-12. tives.Biopolymers, vol. 4, 1966 (pp. 441448) (copy References C te Kay, 6.,Insolubilised Enzymes. Process Biochemistry, UNITED STATES PATENTS 5A1$11St1968 (PP-l 6 (PY? f G1 sumura, et a ontmuous somerizatlon oucc-se I I 2 3 $3 7 by a Column of Glucose Isomerase. Journal of Food574,062 4/1971 i S a e a 68 Science and Technology, vol. 14, N 0. 12,1967 (pp. 539- 4 a s40 'I'X341F8.

FOREIGN PATENTS 10 953,414 3/1964 Great Britain 195116 DAVID NAFF PumaExammer OTHER REFERENCES US. Cl. X.R.

Goldman, et al., Papain Membrane on a Collodion 195 63 DIG 11 Matrix,Science, vol. 150, 1965 (pp. 758760), Q1534. 15

1. A PROCESS OF PRODUCING AN ENZYMATICALLY ACTIVE MEMBRANE WHICH COMPRISES SWELLING A MEMBRANCE FORMED FROM A PROTEIN SELECTED FROM THE GROUP CONSISTING OF COLLAGEN, ZEIN, CASEIN, OVALBUMIN, WHEAT GLUTEN, FIBRINOGEN, MYSOIN, AND MUCOPROTEIN, OR A POLYPEPTIDE SELECTED FROM THE GROUP CONSISTING OF POLYGLUTAMATE, POLYASPRTATE, POLYPHENYLAMINE, POLYTRYROSINE AND COPOLYMER OF LEUCINE AND P-AMINO PHENYLANALINE TO ITS MAXIMUM CAPACITY, AND SOAKING SAID SWOLLEN MEMBRANE IN AN AQUEOUS SOLUTION OF AN ENZYME AT A TEMPERATURE OF 4-10*C. FOR A PERIOD OF 10 HOURS TO 2 DAYS UNTIL SAID ENZYME COMPLEXES BY FORMING COMPLEXING GROUPS SELECTED FROM THE GROUP CONSISTING OF MULTIPLE HYDROGEN BONDS, SALT LINKAGES AND VAN DER WAALS INTERACTIONS WITH SAID MEMBRANE TO FORM AN ENZYME MAMBRANE COMPLEX, AND THEREAFTER DYING SAID ENZYMEMEMBRANE COMPLEX. 